Bacterial homeostasis and survival is critically dependent on defense mechanisms that modify, deactivate, or extrude cytotoxic molecules such as antiseptics and antibiotics, which passively cross the membrane down their concentration gradients. One ubiquitous and highly conserved mechanism entails the expression of polyspecific membrane transporters, referred to as multidrug (MDR) transporters, which harness the Gibbs energy stored in ion electrochemical gradients to power the uphill vectorial clearance of a broad spectrum of cytotoxic molecules. Energy-coupled isomerization of the transporter between multiple intermediates enables alternating access of the substrate binding site from one side of the membrane to the other. Defining the structural elements mediating alternating access and decoding the mechanism of energy conversion in a lipid bilayer-like environment are exciting frontiers in the field and critical for defining transport mechanisms. This proposal will continue support of a productive research program focused on addressing these questions for two families of ion-coupled MDR transporters that have been implicated in clinical drug resistance. Our approach capitalizes on the tool kit of EPR spectroscopy in the context of high resolution structures, is informed by functional studies, and is contextualized through collaborative molecular modeling efforts. Aim 1 seeks to elucidate principles of ion-substrate coupling, identify residues critical for ion and substrate binding, and reveal how specific transporter-lipids interactions shape the energy landscape of conformational changes in two archetypes of the Multidrug and Toxin Extrusion (MATE) family of multidrug transporters. Aim 2 seeks to identify conserved elements of alternating access and ion-substrate coupling for the major facilitator (MFS) family of MDR transporters. We will test a detailed mechanism of ligand-dependent conformational changes, developed in the previous funding period, that integrate ion coupling with specific lipid interactions in the context of a well-established transport model. Together, the two aims will illuminate mechanistic principles for families of transporters implicated in the phenomenon of drug resistance and basic bacterial defense strategies.